1. Field
This disclosure is concerned generally with cell culture systems and specifically with a system adapted to produce and extract biological substances on a continuous basis from continuously dispersed cells without significant damage to the cells.
2. Prior Art The cultivation of selected cells to produce useful substances has been practiced for many centuries. Improvements have been developed over the years and many of these improvements focus on systems or devices adapted for the continuous production of given biological substances from a cell culture. Examples of such continuous systems are described, for example, in U.S. Pat. No. 4,166,768 to Tolbert et al (combined reactor and substance filter unit) and U.S. Pat. No. 4,178,209 to Tolbert et al (also showing a combined cell culture reactor and substance filtering unit). See also recent U.S. Pat. No. 4,639,422 to Geimer et al (cell culture filter apparatus for the continuous cultivation and harvesting of biological products from a dispersion of mammalian cells), Large Scale Cell Culture in Biotechnology by W. R. Arathoon and J. R. Birch, Science, Vol. 232, pp. 1390-1395, 1986 (discussing problems associated with scaling up of mammalian cell production), and A Radial Flow Hollow Fiber Bioreactor for the Large-Scale Culture of Mammalian Cells, by J. P. Tharaken and P. C. Chau, Biotech. & Bioeng., Vol. XVIII, pp. 329-342, 1986.
With the relatively recent discoveries based on recombinant DNA and monoclonal antibody technologies, increasing attention has been directed to providing cell culturing systems that can produce useful biological substances on a continuous basis. In an ideal system, the great majority of the cells are not damaged, are maintained in an optimum environment for expression of the biological substances, the substance yield is acceptable, and the system has a reasonably long and economically useful life. Although many of the cell culture systems disclosed to date achieve one or more of the above goals, it is difficult to achieve all of the above goals simultaneously in a single system. This is especially true for mammalian cell culture systems, because of their known sensitivity, especially to changing environments. Given the increased attention now being directed to mammalian cell lines for the production of both monoclonal antibodies and biological substances based on recombinant DNA techniques, the achievement of all of the above goals in a single mammalian cell culture system is highly desirable.
Although the scale up of mammalian cells still is considered difficult, several additional cell culture systems have been proposed. To simultaneously reach high cell densities and harvest the product released into the cell culture medium, systems are available that in one way or the other suspend or immobilize the cells to be cultured. Hollow-fiber cartridges for suspended cells (see European Patent Application No. 0 112 155, assigned to BioResponse, Inc.) as well as ceramic cartridges for immobilized cells (see B. G. D. Bodeker et al, Develop. Biol. Standard, Vol. 66, pp. 473-479 [S. Karger, Basel, 1987]) are now used for suspended cells or to immobilize cells which are then perfused with the cell culture medium. An advantage of these systems is that product can be harvested from a medium that contains only a low number of cells or no cells at all.
Several attempts have been made to adapt the deep tank fermentor technology used for microbial fermentation to the cultivation of mammalian cells. Both anchorage dependent and independent cells have been grown in deep tank fermentors. Amongst others, however, one key problem is to establish a continuous culture. Traps and spinning filters have been used to separate the product containing medium from the cells and to allow a long term cultivation process. Unfortunately, however, both processes have been shown to be less efficacious than expected. The spinfilters tend to clog and afterwards do not allow medium to penetrate. Such high density reactors are therefore difficult, if not impossible, to use with existing technology.
The approach described in this disclosure avoids many of the above problems. In one embodiment, the system of this disclosure limits the amount of dispersed cell culture and its duration outside of the reactor. A preferred system also facilitates the continuous dispersion of the cells by continuously breaking up cell aggregates and uses an external filtering device such as a hollow fiber membrane with having a controlled average pore size (e.g. 0.6 um pore size) to separate the substance to be harvested with the medium from the circulating cells. The outside-the-reactor placement of the filter or porous hollow fiber device permits control over the volume of culture outside the reactor at any one time and the length any one dispersed cell is outside the reactor. The device is located outside of the fermentor and can be easily replaced when the membrane is clogged and penetration of substance and medium is no longer obtained. Details of our system are described below.